Acute lung injury (ALI) and the Adult Respiratory Distress Syndrome (ARDS) claim approximately 75,000 lives in North America each year. While mechanical ventilation is a life saving treatment, it may nevertheless cause harm by straining lungs at a time they are particularly prone to injury from deforming stress. Unlike a broken bone which can be casted, one cannot immobilize injured lungs. Therefore, even so-called lung protective ventilator settings may not spare them from further damage. For the past three years we have studied the determinants of cellular stress failure as a central driver of the lungs' innate immune response to deforming stress. We have shown that cells of lungs, which are exposed to mechanical ventilation with high volumes and trans-pulmonary pressure, experience reversible plasma membrane wounds and that such wounds can trigger proinflammatory signaling cascades. In this competing renewal application we will identify and test therapies designed to protect alveolar epithelial cells from deformation injury or to promote wound repair and we will define their mechanisms of action. Guided by compelling preliminary data we will focus our efforts on osmotic stress as lung-protective intervention and test the efficacy of concentrated salt and sugar solutions in experimental models of ventilator induced lung injury. The proposed experiments will serve three specific aims: 1) To test the effect of osmotic stress on the susceptibility of lung cells to deformation injury and on the probability of cell repair; 2) To examine the effect of osmotic stress on the regulation of volume, surface area and plasma membrane tension in type I and type II alveolar epithelial cells (AEC); 3) To examine the importance of purinergic signaling on osmotic stress mediated alveolar epithelial repair. Experiments will be carried out on anesthetized mechanically ventilated rodents, in isolated perfuse lungs and on cells that will be injured and/or deformed in tissue culture. Study endpoints include the number of wounded and/or repaired cells as assessed by live specimen microscopy, measures of lung inflammation, as well as measures of lung and cell structure and function. We will use optical traps to characterize the length tension relationships of plasma membrane tethers in AEC's and interpret the data as a biophysical readout of membrane area and tension regulation and the effects of osmotic stress on them. The knowledge we will gain, will inform about disease mechanisms and the risks and benefits of conducting a clinical trial. PUBLIC HEALTH RELEVANCE. Our group investigates the determinants of cellular stress failure as a central driver of the lungs' innate immune response to deforming stress. In preliminary experiments we have identified osmotic stress as potential cytoprotective adjunct to mechanical ventilation. The proposal seeks to establish the efficacy of this intervention in preclinical models of ventilator induced lung injury and to dissect its mechanisms of action. [unreadable] [unreadable] [unreadable]